WAKFlow® HLA Typing kit Instruction manual
Contents
1. Biotin gt PCR 1 5 hours tM Amplification of target genes with biotinylated locus specific primers Denaturation 5 minutes A 1 Denaturation of the amplified DNA into single strand Neutralization Hybridization and fluorescent labeling PCR amplicon 30 minutes oligonucleotide probe microshere bead Hybridization of denatured DNA to oligonucletide probes immobilized on microshpere beads and fluorescent labeling with SAPE in a single step Washing 5 minutes Data acquisition and analysis with the Luminex system 3 4 WAKFlow HLA typing kit protocol 1 Preparation of sample DNA in the pre PCR area Sample DNA should be prepared in a pre PCR area to avoid PCR contamination Recommended concentration of sample DNA is 20 ng L Adjust concentration with nuclease free water if necessary 2 PCR in the pre PCR area Note Wear lab coat and gloves dedicated for the pre PCR area to avoid PCR contamination Enter the PCR program below into your thermal cycler Turn on the thermal cycler to warm up heated lid Mix thoroughly PCR Pre mix before use 2 1 Prepare the appropriate amount of the following reaction mixture Components of a 1x PCR mixture PCR Pre mix specific for each HLA locus DNA Polymerase Soln specific for each HLA locus Note Preparation of PCR reaction should be done on ice to avoid non specific gene amplification 22. 2 Aliquot 25 wL of the PCR reaction mixture of step 2 1 into a PCR reaction tube or a 96 well PCR plate then add 2 u L of sample DNA prepared in step 1 Note Change pipette tip for each sample DNA lt 2 3 Cap or seal PCR tubes or PCR plate and place samples in a thermal cycler then run the program below 93 C 3min l 93 C 30 sec 60 C 30sec a 40 cycles 72 C 30sec l 4 C Hold Note This PCR reaction may take about 1 5 hours when utilizing the GeneAmp PCR System 9700 Gold 96 well For another type of thermal cycler it may be necessary to optimize the PCR cycle empirically For the GeneAmp PCR System 9700 Gold 96 well set Reaction volume to 27 u L and Ramp Speed to MAX mode on the Selection Method Options window before starting the PCR reaction The reaction mixture should be stored at 4 C when it does not proceed to hybridization process immediately after PCR Storage at room temperature may cause low signals 3 Hybridization and data acquisition Note Wear lab coat and gloves dedicated for the post PCR area Turn on a thermal cycler and run the program of 55 C HOLD Turn on the Luminex system set temperature of XY Platform to 37 C and perform the warm up procedure Make sure that the Denaturation soln Hybridization soln and Wash soln are at room temperature when used Denaturation soln is an alkaline reagent Wear protective glasses and gloves 3 1 Aliquo
3. PCR contamination Contamination of previously amplified DNA into PCR reaction may cause incorrect typing results To avoid PCR contamination Separate pre PCR area from post PCR area Pre and Post PCR area must have their own separate set of lab coat shoes gloves and equipment including micropipettes pipette tips racks thermal cycler and so on These items should not leave the area to which they are assigned After PCR preparation wipe the lab bench with 0 5 sodium hypochloride solution or bleach diluted ten fold with H20 3 2 Instrument and equipment requirements 1 PCR in the pre PCR area Thermal cycler Micropipette 2 20 u L 20 200 u L 200 1000 u L Sterilized micropipette tips Sterilized 0 2mL PCR tubes or 96 well PCR plate Gold 96well GeneAmp PCR System 9700 Micropipett 2 Hybridization in the post PCR area Multichannel micropipette Micropipette 2 20 u L 20 200 u L 200 1000 u L Sterilized micropipette tips Repetitive micropipette Vortex mixer Sterilized 96 well low profile PCR plate Centrifuge with swing bucket rotor for 96 well microtiter plate Multichannel micropipette Repetitive micropipette Vortex mixer Centrifuge 3 Data acquisition in the post PCR area Luminex system Microsoft Windows 2000 or XP personal computer with Intel Pentium lll 500MHz processor Recommended Luminex System 3 3 Procedure of the WAK Flow HLA typing kit
4. Revised in Aug 2008 R Issued in Jun 2005 FAKUN AGA WAK Flow HLA Typing kit Instruction manual Research use only 4 Edition oY Wakunaga Pharmaceutical Go Ltd Contents 1 Principles of HLA TY GING ethecver seu cetecoceuictet et tse cecueccess 2 Kit components and storage CONGItIONS cceeeeeeeeeeeeeeeees 30 PMOLOCOIS vacceactenacsassidduanatawhtdetaddcvitheddseaesteoabhe E ET EEL AA aiara i 3 1 Attentions to avoid PCR contamination 2 3 2 Instrument and equipment requirements c0006 2 3 3 Procedure of the WAK Flow HLA typing kit 4 3 4 WAK Flow HLA typing kit protocol ccccceeeeees 5 1 Preparation of sample DNA 2 PCR 3 Hybridization and data acquisition 4 Data analysis 4 Warning or CAUTIONS iinien ce ccslaah oe ethaansielnes ni woos lvaesddicthesees 1 Principles of HLA typing The WAKFlow HLA typing kit is based on the reverse sequence specific oligonucleotide probes SSO method coupled with xMAP technology designed for use with the Luminex system http Awww luminexcorp com to identify HLA alleles encoded by sample DNA The target DNA is first amplified by polymerase chain reaction PCR with biotinylated primers specifically designed for each HLA locus The PCR product is denatured and hybridized to complementary oligonucleotide probes immobilized on fluorescently coded microsphere beads At the same time t
5. he biotinylated PCR product is labeled with phycoerythrin conjugated streptavidin to allow it to be detected by the Luminex system The entire process is performed in a single well of a 96 well PCR plate thus 96 samples can be treated at one time The HLA alleles are assigned by analysis of the reaction hybridization pattern of the target sample using the WAK Flow HLA typing software 2 Kit components and storage conditions Product Size WAKFlow HLA typing kit 96 tests kit Amplification Package Store below 15 C PCR pre mix specific for each HLA locus tttttsssese 1 323 u L 2 tubes DNA Polymerase Soln specific for each HLA locus s tttttttttts 54 uL 1 tube Detection Package Store at 2 8 C Denaturation Soln ree ee ee ee ee ee ee 1 000 u L 1 tube Hybridization Soln were Pee eee eee ee ee ee ee ee 2 2 200 u L 1 tube Beads mix specific for each HLA locus ssss ss ssususssunu 330 uL 1 tube SAPE Phycoerythrin conjugated Streptavidin s s s s 220 uL 1 tube D Wash Soln sasuonnsnsoaonnasnonnnssanasononassosonssonnnnnsonasnonn 50 mL 1 tube Plate Sealing Sheet eee eee ee ee ee 2 sheets Note PCR Pre mix and DNA polymerase soln should be stored below 15 C for longer storage 6 Beads mix and SAPE must be stored at 2 8 C under dark conditions Do not mix components from different lots or products 3 Protocols 3 1 Attentions to avoid
6. ndex html and updated in January every year 4 Warning or caution This product is for research use only Do not use for the diagnosis and prognosis of disease Do not use expired reagents Denaturation Soln and Hybridization Soln are corrosive and may cause burns In case of contact with reagents immediately wash the eyes or skin with a large amount of water Call a physician in case of burns inhalation ingestion and so on 4 Pay attentions to PCR contamination This may cause incorrect typing results For the detail see section 3 1 Attentions to avoid PCR contamination 5 The specifications may be changed to improve the kit without notice Effective period 12 months indicated on product box Packaging unit gt 96 tests kit Manufactured by gt ae Wakunaga Pharmaceutical Co Ltd 1624 Shimokotachi Koda cho Akitakata city Hiroshima 739 1195 Japan E Mail wakunaga hla wakunaga co jp URL http Awww wakunagahla jp
7. supernatant by snapping Note More than 5uL of supernatant remaining after snapping may lead to high background signal Additional washing step may be effective to lower the background signal 3 7 Add 75 uL of Wash soln to each well 3 8 Make sure that XY Platform is set at 37 C Eject the plate stage from the Luminex XY Platform place the PCR plate in step 3 7 and then retract the stage ea 3 9 Choose a template file corresponding to the roduct lot enter number of samples and then start data acquisition 4 Data acquisition and analysis Note Data acquisition should be carried out immediately Store the samples under dark condition at 37 C if they are not measured immediately It will be cause of non specific hybridization if XY Platform and sample plate are left at room temperature 4 1 Open an output CSV_file which is automatically created after data acquisition with WAKFlow typing software and perform genotyping Positive or negative reaction is assigned based on the pre set cut off value for each typing probe A fluorescent signal higher than the cut off value is defined as a positive reaction HLA alleles are assigned automatically by matching the pattern of positive and negative reactions of each bead HLA alleles used for the WAKFlow HLA typing kit are cited from the report of the Anthony Nolan Research Institute HLA Informatics Group http www anthonynolan org uk HIG i
8. t 5 u L of Denaturation Soln into a 96 well PCR plate r 3 2 Add 5 u L of PCR product of step 2 3 into each well then mix thoroughly b ipetting or vortex and incubate at room temperature for 5 10 minutes 3 3 Prepare an appropriate amount of the following hybridization mixture in a sample tube and mix thoroughly by vortex Components of a 1x hybridization mixture Hybridization Soln Beads mix specific for each HLA locus Note Suspend the Beads mix thoroughly by vortex before use 3 4 Add 25 u L of the hybridization mixture prepared in the ste 3 3 to the denatured DNA from step 3 2 Seal the 96 well PCR plate tightly with a_plate sealing sheet and mix thoroughly by vortex Note Apply of the hybridization mixture should be done quickly within 3 minutes because the Beads mix and SAPE are light sensitive Sealing of the PCR plate should be done thoroughly to avoid well to well sample contamination After mixing the PCR plate collect the hybridization mixture to the bottom of the PCR plate by snapping 3 5 Place the 96 well PCR plate onto the thermal cycler pre warmed to 55 C tighten the lid and then incubate for 30 minutes Note Make sure that the Luminex system has been warmed up and ready for measurement of fluorescent signal 3 6 Add 75uL of Wash soln to each well then centrifuge the plate for 1 minute at 3 000 rom approx 1000x g Remove
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